Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291.

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio.

Crosslinking kiwifruit-derived DNA with natural aromatic aldehydes generates membranolytic antibacterial nanogels.

Improper use of antibiotics has led to the global rise of drug-resistant biofilm bacteria. Thus, researchers have been increasingly interested in green materials that are highly biocompatible and have low toxicity. Here, nanogels (NGs) with imine bonds were synthesized by crosslinking kiwifruit-derived DNA's primary amine and aromatic aldehydes (cuminaldehyde, p-anisaldehyde, or vanillin) under water-in-hexane emulsion processes. Transmission electron microscope showed that the NGs had spherical geometry with an average particle size ranging from 40 to 140 nm and that the zeta potential indicated a negative charge. Additionally, the DNA-aromatic aldehyde NGs showed low cytotoxicity toward normal cell organoids and human RBCs in cell viability tests. These NGs were also tested against four pathogenic bacteria for various assays. DNA-vanillin (DNA-VA) NGs exhibited significant antibacterial effects against bacteria with very low inhibitory concentrations as seen in a minimum inhibitory concentration assay. Scanning electron microscope observation revealed that the bacteria were deformed, and immunoblotting detected intracellular groEL protein expression. In agreement with these results, DNA-aromatic aldehyde NGs successfully protected C. elegans from P. aeruginosa-induced lethality. These DNA NGs provided a multivalent 3D space for antibacterial aromatic aldehydes to tether, enhancing their interaction with the bacterial wall. These results offer a new direction for the development of novel antibiotics in the future.

Natural nanogels crosslinked with S-benzyl-L-cysteine exhibit potent antibacterial activity.

Biofilm-forming bacteria E. coli and P. aeruginosa have both exhibited resistance against multiple antibiotics in clinical settings. To find a solution, researchers have turned to antibacterial structurally modified from natural materials that are harmless to the human body. Among these is DNA, a natural polymer composed of deoxyribose that when treated with HCl exposes its aldehyde groups and produces DNA-HCl. Here, we crosslinked these aldehyde groups with the primary amines in S-benzyl-L-cysteine (SBLC) using a Schiff reaction to obtain DNA-HCl-SBLC. We additionally treated alginate acid (AA) with EDAC, obtaining AA-EDAC, and substituting it with SBLC to produce AA-SBLC. We incorporated the above reactions with an emulsification process to produce nanogels (NGs) that were verified to be spherical and possessing benzene rings successfully grafted onto DNA-HCl and AA-EDAC. These natural NGs were proven to be negatively charged through zeta potential analysis and presented low cytotoxicity toward normal cells in cell organoid viability assays. These SBLC-modified polymers provided better inhibition of bacterial growth than those without modification. Moreover, after incubation with SBLC-modified NGs, bacteria expressed intracellular recA or pvdA in a dose-dependent manner, which was consistent with SEM data from damaged bacteria. Out of four tested NGs, DNA-HCl-SBLC NGs suppressed P. aeruginosa-induced sepsis most effectively and extended the lifespan of C. elegans. This study provides an alternative clinical solution to antibiotics-resistant biofilm strains.

Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

A multi-omic analysis reveals the role of fumarate in regulating the virulence of enterohemorrhagic Escherichia coli.

The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Several molecular mechanisms have been described for the pathogenicity of EHEC; however, the role of bacterial metabolism in the virulence of EHEC during infection in vivo remains unclear. Here we show that aerobic metabolism plays an important role in the regulation of EHEC virulence in Caenorhabditis elegans. Our functional genomic analyses showed that disruption of the genes encoding the succinate dehydrogenase complex (Sdh) of EHEC, including the sdhA gene, attenuated its toxicity toward C. elegans animals. Sdh converts succinate to fumarate and links the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) simultaneously. Succinate accumulation and fumarate depletion in the EHEC sdhA mutant cells were also demonstrated to be concomitant by metabolomic analyses. Moreover, fumarate replenishment to the sdhA mutant significantly increased its virulence toward C. elegans. These results suggest that the TCA cycle, ETC, and alteration in metabolome all account for the attenuated toxicity of the sdhA mutant, and Sdh catabolite fumarate in particular plays a critical role in the regulation of EHEC virulence. In addition, we identified the tryptophanase (TnaA) as a downstream virulence determinant of SdhA using a label-free proteomic method. We demonstrated that expression of tnaA is regulated by fumarate in EHEC. Taken together, our multi-omic analyses demonstrate that sdhA is required for the virulence of EHEC, and aerobic metabolism plays important roles in the pathogenicity of EHEC infection in C. elegans. Moreover, our study highlights the potential targeting of SdhA, if druggable, as alternative preventive or therapeutic strategies by which to combat EHEC infection.

HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans.

Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.